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Simple Summary  The biodiversity and ecological status assessment of transitional water ecosystems by benthic macroinvertebrates investigation could use DNA barcode tools for more rapid and efficient outputs. The principal limits of this application are the incompleteness of DNA barcode databases, the identification of optimal primers set, and the gap in the species sequences. The influence of the incompleteness of DNA barcode libraries on species diversity indices, ecological indicators, and ecological status assessment in transitional waters of the southeast Mediterranean were analysed, underlying the importance to implement DNA barcode libraries and to put an effort toward specific species at a local level.    Abstract  The ecological assessment of European aquatic ecosystems is regulated under the framework directives on strategy for water and marine environments. Benthic macroinvertebrates are the most used biological quality element for ecological assessment of rivers, coastal-marines, and transitional waters. The morphological identification of benthic macroinvertebrates is the current tool for their assessment. Recently, DNA-based tools have been proposed as effective alternatives. The main current limits of DNA-based applications include the incompleteness of species recorded in the DNA barcode reference libraries and the primers bias. Here, we analysed the influence of the incompleteness of DNA barcode databases on species diversity indices, ecological indicators, and ecological assessment in transitional waters of the southeast Mediterranean, taking into account the availability of commonly sequenced and deposited genomic regions for listed species. The ecological quality status assigned through the potential application of both approaches to the analysed transitional water ecosystems was different in 27% of sites. We also analysed the inter-specific genetic distances to evaluate the potential application of the DNA metabarcoding method. Overall, this work highlights the importance to expand the barcode databases and to analyse, at the regional level, the gaps in the DNA barcodes.

Testing the Influence of Incomplete DNA Barcode Libraries on Ecological Status Assessment of Mediterranean Transitional Waters